CN1820067A - Nucleic acid amplifier and method of nucleic acid amplification - Google Patents
Nucleic acid amplifier and method of nucleic acid amplification Download PDFInfo
- Publication number
- CN1820067A CN1820067A CN200480019643.5A CN200480019643A CN1820067A CN 1820067 A CN1820067 A CN 1820067A CN 200480019643 A CN200480019643 A CN 200480019643A CN 1820067 A CN1820067 A CN 1820067A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- stream
- template
- renaturation
- zone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
- B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/087—Multiple sequential chambers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
- B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
- B01L2300/1827—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using resistive heater
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1861—Means for temperature control using radiation
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
Abstract
A nucleic acid amplifier capable of efficiently performing PCR even with the use of nucleic acid synthetase lacking heat resistance, which nucleic acid amplifier can realize nucleic acid synthetase reutilization, scaleup, etc. and facilitates separation/purification of nucleic acid after amplification; and a method of nucleic acid amplification performed therewith. In particular, the method comprises causing at least a nucleic acid as a template, a nucleic acid as a primer and a reaction solution containing a phosphate compound and a metal ion to flow through a channel having a region for melting an intramolecularly and/or intermolecularly formed double strand of nucleic acid so as to effect denaturation into single strands and a regeneration region for regenerating a double strand of nucleic acid from the nucleic acid after the double strand melting, thereby melting at the denaturation region the intramolecularly and/or intermolecularly formed double strand of nucleic acid as the template so as to effect denaturation into single strands and thereafter at the regeneration region not only regenerating a double strand between the template nucleic acid after the double strand melting and the primer nucleic acid but also carrying out nucleic acid synthesis with the nucleic acid synthetase fixed within the regeneration region.
Description
Technical field
The present invention relates to use the nucleic acid amplifier and the nucleic acid amplification method of PCR method, more specifically relate to and have at least one stream in inside, in this stream, import the reaction solution contain nucleic acid, nucleic acid, phosphate cpd and metal ion at least, the nucleic acid amplifier and the nucleic acid amplification method that carry out the amplification of nucleic acid by the nucleic acid synthetic enzyme that is fixed in this stream as primer as template.
Background technology
PCR (polymerase chain reaction) method be widely used in trace template DNA duplicate amplification efficiently.The PCR method is by repeatedly repeating the increase method of target dna of following circulation, and its 1 circulation comprises: will become the step of single stranded DNA as the double-stranded DNA thermally denature of template; On the single-stranded template DNA that obtains respectively with the step of complementary primer annealing; By having stable on heating archaeal dna polymerase effect, carry out complementary strand by primer and synthesize, thus the step of synthetic dsdna.
Above steps was undertaken by the temperature and the reaction times of control reaction solution, usually, double-stranded DNA thermally denature as template becomes single stranded DNA to carry out under about 94 ℃, on the single stranded DNA primer annealing is being carried out under about 55 ℃, by carrying out the synthesizing under about 72 ℃ of complementary strand of archaeal dna polymerase.
In the past, as the device that automatically performs the PCR method, known have a device that reacts by the following method: the reaction solution that will contain template DNA, primer, dNTP, archaeal dna polymerase etc. adds in the Eppendorf type test tube, this test tube is inserted in the hole that is arranged on the aluminium part, change the temperature of this aluminium parts with well heater and water cooler.
Yet above-mentioned PCR method need be carried out thermal cycling under the control of high-precision temperature, as in the above-mentioned batch-wise reaction along with the increase of scale, the fluctuation of the heat of reaction system becomes significantly, so there is the limit in the lifting of scale.
Therefore, as under the control of high-precision temperature, carrying out thermal cycling and can promoting the PCR method of amplification amount, in following patent documentation 1 and following non-patent literature 1, disclosed streaming PCR method.This streaming PCR method is to carry out thermal cycling by the reaction solution that contains archaeal dna polymerase, template DNA, primed DNA, dNTP etc. to the stream input that is provided with heating part and cooling end, thereby carries out the method for PCR.
In addition, disclosed the amplification method of following nucleotide sequence in the following patent documentation 2, it is the method that is used for amplifying nucleic acid sequence, it is characterized in that, comprise: (a) will handle by archaeal dna polymerase with at least a kind of primer of the actual complementary of the base sequence of this nucleic acid, and synthesize step with this template complementary primer extension chain as the nucleic acid of template; Wherein, this primer is the mosaic oligonucleotide primer that contains deoxyribonucleotide and ribonucleotide, and in order to shear with endonuclease, this ribonucleotide is configured in 3 ' terminal or a 3 ' terminal side of this primer; (b) with the position of the containing ribonucleotide endonuclease cutting steps of the primer extension chain of the double-strandednucleic acid that obtains in (a) step; (c) quilt that obtains in (b) step cuts the primer 3 ' end partly of the double-strandednucleic acid of primer extension chain, make and the extension of template complementary nucleotide sequence by archaeal dna polymerase with chain transfer activity, thus the step of carrying out chain transfer.If adopt this method (ICAN method), can not carry out thermal cycling and DNA amplification does not have stable on heating enzyme so can use, and do not have the restriction of the reaction scale that the fluctuation of heat causes.
Patent documentation 1: Japanese patent laid-open 6-30776 communique
Patent documentation 2: the Japanese Patent spy opens the 2003-70490 communique
Non-patent literature 1: No. 280 5366 volumes in " Science " (1998), 1046-1048 page or leaf (Kopp MU, Mello AJ, Manz A. work)
The announcement of invention
The problem to be solved in the present invention
Yet, above-mentioned batch-wise PCR method, above-mentioned patent documentation 1 and above-mentioned non-patent literature 1 described PCR method need heating when making double-stranded template DNA sex change for single stranded DNA, therefore need to use to have stable on heating special archaeal dna polymerase, existence can't be used the ubiquitous shortcoming that does not have stable on heating archaeal dna polymerase.
In addition, in the method that above-mentioned patent documentation 2 is disclosed, the mosaic primer that uses RNA and DNA is as primer, need use the exo-Bca archaeal dna polymerase of synthetic DNA when the dna double chain unwinds and the special enzymes such as ribonuclease H of the tie point of DNA that shears new extension on the mosaic primer and mosaic primed RNA, so the shortcoming that exists cost to raise.
In addition, be mixed with nucleic acid synthetic enzyme such as archaeal dna polymerase in the above-mentioned method in the past in the resultant of reaction, therefore not only the purifying of the DNA of amplification bothers, and expensive nucleic acid synthetic enzyme almost can't utilize again.
Therefore, have stable on heating nucleic acid synthetic enzyme and use and do not have stable on heating nucleic acid synthetic enzyme and also can carry out PCR continuously, efficiently even the object of the present invention is to provide to be not only to use, the nucleic acid synthetic enzyme can utilize again, use continuously, and the separation and purification of the nucleic acid of amplification is easy, can also promote the nucleic acid amplifier and the nucleic acid amplification method of scale.
Solve the method for problem
In order to reach above-mentioned purpose, nucleic acid amplifier of the present invention is to have at least one stream in inside, in this stream, flow through the reaction solution of the nucleic acid, nucleic acid, phosphate cpd and the metal ion that contain at least as template, the nucleic acid amplifier of amplification of nucleic acid in this stream as primer; It is characterized in that, described stream has denatured areas and renaturation zone simultaneously, the reaction of degeneration that the two strands of the intramolecularly of described nucleic acid as template and/or intermolecular formation is unwind, what make in the described renaturation zone that described two strands unwinds forms two strands as the nucleic acid of template and described nucleic acid as primer, and the nucleic acid synthetic enzyme is fixed in described renaturation zone.
If adopt nucleic acid amplifier of the present invention, when the reaction solution that then imports the nucleic acid that contains at least as template, nucleic acid, phosphate cpd and metal ion as primer in having at least one the stream in above-mentioned denatured areas and renaturation zone carries out the nucleic acid building-up reactions, the above-mentioned nucleic acid synthetic enzyme that is fixed on above-mentioned renaturation zone can not be subjected to making as the nucleic and melting of template and the influence of sex change heating when becoming the strand state etc., so the inactivation of nucleic acid synthetic enzyme is suppressed,, use can not carry out PCR continuously even not having stable on heating nucleic acid synthetic enzyme yet.In addition, above-mentioned nucleic acid synthetic enzyme is a fixed, thus the separation and purification of the nucleic acid that not only can easily increase, and also above-mentioned nucleic acid synthetic enzyme can utilize again, use continuously, and the lifting of reaction scale also is easy.Among the present invention, mention under the situation of nucleic acid, its implication comprises two class nucleic acid of natural type and non-natural type.
In the nucleic acid amplifier of the present invention, better be to have the temperature control unit that can heat above-mentioned denatured areas and above-mentioned renaturation zone be remained on the temperature lower than above-mentioned denatured areas.If adopt this mode, can carry out a series of PCR circulation that is made of following steps continuously, efficiently: make the nucleic and melting as template that has in intramolecularly and/or the intermolecular two strands that forms by heating, sex change is the step of strand state; Described than the low environment of the temperature of denatured areas under, this two strands of obtaining unwind as the nucleic acid of template and form double-stranded step respectively between the complementary primer; Described than the low environment of the temperature of denatured areas under, make itself and the effect of nucleic acid synthetic enzyme, carry out complementary strand synthetic step by primer.
In addition, better be that described nucleic acid synthetic enzyme is fixed on the bead, this bead is filled in the described renaturation zone at least.If adopt this mode, fixed nucleic acid synthetic enzyme is contacted efficiently, with reaction solution so can improve reaction efficiency.
Perhaps, above-mentioned nucleic acid synthetic enzyme also can be fixed on the inner wall surface in described renaturation zone at least.If adopt this mode, can form the stream of immobilized nucleic acids synthetic enzyme simply.That is, when forming such stream, at first can make the nucleic acid synthetic enzyme be immobilized in the whole surface of stream and form overall flow paths, in this way, even the enzyme deactivation of denatured areas, the enzyme in renaturation zone still keeps active condition, so can easily form required stream.
In addition, better be that above-mentioned denatured areas and above-mentioned renaturation zone alternatively are set in above-mentioned stream.If adopt this mode, then can carry out repeatedly the PCR circulation, so the target nucleic acid that can increase efficiently.
In the nucleic acid amplifier of the present invention, above-mentioned nucleic acid synthetic enzyme can use the enzyme that has optimum temperuture 30～40 ℃ temperature.If adopt this mode, then can enlarge the range of choice that the nucleic acid synthetic enzyme uses, the enzyme of the less expensive that can't use among the selection PCR in the past.In addition, can also use other enzyme except that general nucleic acid synthetic enzyme simultaneously.Therefore, the enzyme that was difficult to use simultaneously when in the past carrying out PCR by using simultaneously, for example correct the enzyme etc. of the mispairing of the nucleic acid that has synthesized, comparable PCR in the past improves the reliability of amplification.
In addition, in the nucleic acid amplifier of the present invention, above-mentioned stream can have the circulation stream, and should the circulation stream has the structure of above-mentioned renaturation zone and above-mentioned denatured areas.
Here, the circulation stream is meant and is used to make reaction solution to circulate, also alternately pass through the interior denatured areas of this circulation stream and the stream in renaturation zone, the stream that the stream that to be above-mentioned stream interconnected again at position branch, this ramose stream of regulation, have ring texture or the integral body of above-mentioned stream have one or more ring texturees utilizes part or all of flow path liquid of this ring texture to flow through ring texture and circulates.
If adopt this mode, then can make nucleic acid as template, as the nucleic acid of primer, phosphate cpd etc. in the internal recycle of designated area, flow through denatured areas, renaturation zone repeatedly, so can prevent template uses up, and utilize reaction solution energetically again, therefore can lower running cost.
In addition, nucleic acid amplifier of the present invention better is to have the liquid feed device that the control reaction solution flows to, and the flow direction of this liquid feed device control reaction solution periodically reverses.If adopt this mode, then can use various in limited volume the liquid feed device of feed flow, the simplification of liquid feed device is become easily, with the miniaturization of adaptive device.
In addition, nucleic acid amplification method of the present invention is to be used for containing nucleic acid as template, the reaction solution amplification of nucleic acid, phosphate cpd and metal ion as primer at least as the method for the nucleic acid of template, it is characterized in that, comprise: the denaturing step that the two strands of the intramolecularly of described nucleic acid as template and/or intermolecular formation is unwind; (b) with step (a) different zone, this two strands that obtains in the step (a) unwind as the nucleic acid of template and described as forming double-stranded renaturation step between the nucleic acid of primer; (c) in step (b) and/or after, make to be fixed in the step that the nucleic acid synthetic enzyme zone that comprises the zone of carrying out step (b), that keep active condition contacts with reaction solution.
If adopt nucleic acid amplification method of the present invention, then can carry out above-mentioned denaturing step and above-mentioned renaturation step respectively in different zones, the influence of heating when the above-mentioned nucleic acid synthetic enzyme that is fixed in the zone that comprises the zone of carrying out the renaturation step can not be subjected to making as the nucleic acid denaturation of template etc., so the inactivation of nucleic acid synthetic enzyme is suppressed,, use can not carry out PCR continuously even not having stable on heating nucleic acid synthetic enzyme yet.In addition, above-mentioned nucleic acid synthetic enzyme is a fixed, thus the separation and purification of the nucleic acid that not only can easily increase, and also above-mentioned nucleic acid synthetic enzyme can utilize again, use continuously, and the lifting of reaction scale also is easy.
The effect of invention
If adopt the present invention, then the two strands with the intramolecularly that makes nucleic acid and/or intermolecular formation unwind and sex change to be the zone of strand state and nucleic acid that this two strands has been unwind form in the stream in double-stranded renaturation zone once more, import the nucleic acid that contains at least as template, nucleic acid as primer, when the reaction solution of phosphate cpd and metal ion carries out the nucleic acid building-up reactions, the influence of heating when the above-mentioned nucleic acid synthetic enzyme that is fixed in the renaturation zone can not be subjected to making as the nucleic acid denaturation of template etc., so the inactivation of nucleic acid synthetic enzyme is suppressed,, use can not carry out PCR continuously even not having stable on heating nucleic acid synthetic enzyme yet.In addition, above-mentioned nucleic acid synthetic enzyme is a fixed, thus the separation and purification of the nucleic acid that not only can easily increase, and also above-mentioned nucleic acid synthetic enzyme can utilize again, use continuously, and the lifting of reaction scale also is easy.
The simple declaration of accompanying drawing
The synoptic diagram of a kind of embodiment of [Fig. 1] nucleic acid amplifier of the present invention.
The partial mode figure of the stream of [Fig. 2] above-mentioned nucleic acid amplifier.
The synoptic diagram of the another kind of embodiment of [Fig. 3] nucleic acid amplifier of the present invention.
The mode chart of the circulation stream of [Fig. 4] above-mentioned nucleic acid amplifier.
The explanatory view of temperature control unit is used in [Fig. 5] above-mentioned sex change that is used to form denatured areas and renaturation zone with temperature control unit and renaturation.
The synoptic diagram of another embodiment of [Fig. 6] nucleic acid amplifier of the present invention.
Be immobilized in the mode chart of the nucleic acid synthetic enzyme in the kapillary in [Fig. 7] same nucleic acid amplifier.
1 unitary mode chart of the stream of used nucleic acid amplifier in [Fig. 8] embodiments of the invention.
The result's that [Fig. 9] detects by same nucleic acid amplifier amplification of nucleic acid and with agarose gel electrophoresis photo.
The explanation of symbol
1,1a～1g substrate
2 streams
The 2a filling orifice
2b takes out the hole
2c branch stream
3 bead filling parts
The 3a wide diameter part
4 beads
5 nucleic acid synthetic enzyme
6 immobilized nucleic acids synthetic enzyme
10,20,50 nucleic acid amplifiers
11～13, the outside liquid feed device of 13a
14 the 1st reaction liquid baths
15 the 2nd reaction liquid baths
16 reaction liquid baths
31,32 thermostatic baths
33,52 temperature-control devices
34,39 sex change temperature control units
35,40 renaturation temperature control units
36,37 stirrers
38 dividing plates
51 kapillaries
A sex change temperature province
B renaturation temperature province
The best mode that carries out an invention
At first, nucleic acid amplification method of the present invention is described.
In the nucleic acid amplification method of the present invention, by at least one stream, import the nucleic acid (hereinafter to be referred as template) contain at least as template, as the reaction solution of nucleic acid (hereinafter to be referred as primer), phosphate cpd and the metal ion of primer, in described stream, carry out described template sex change, sex change the described template and the annealing of described primer, nucleic acid synthetic of using the nucleic acid synthetic enzyme; Be provided with in the described stream denatured areas make the reaction of degeneration that has formed double-stranded described nucleic acid as template and become the strand state described as template nucleic acid and the annealing reaction of described primer in carry out the renaturation zone of nucleic acid building-up reactions, fixed nucleic acid synthetic enzyme at least a portion of the stream in described renaturation zone by the nucleic acid synthetic enzyme.The sex change of template is to instigate to form double-stranded nucleic and melting, becomes the state of strand.
Above-mentioned denatured areas is set at the required environment of template sex change, for example is set at 1) melting temperature(Tm) that is set in nucleic acid is above, 2) be set at alkalescence acid, 3) do not contain positively charged ion, 4) sneak into the environment of hydrogen bond inhibitor (for example urea and guanidinesalt etc.) etc.
Among the present invention, for sex change and the renaturation that repeats nucleic acid, in above-mentioned condition, but better be repeatedly setting to be set in the melting temperature(Tm) of nucleic acid above or to be set at alkalescence acid, it is most effective being set in more than the melting temperature(Tm) of nucleic acid, so good especially.For example, the sex change of template is heated more than the melting temperature(Tm) of nucleic acid and when carrying out, and is different with sequence according to the length of template, cannot treat different things as the same, and for example can be heated to 90～99 ℃ under the situation of the template of hundreds of base pairs, better be 92～97 ℃.
Owing to be difficult to give nucleic acid synthetic enzyme alkali resistance, so in the PCR method in the past, do not use the sex change condition of alkaline environment as template, but among the present invention, the zone of fixed nucleic acid synthetic enzyme can be set at neutral environment, if be set at neutral environment like this, then also denatured areas can be set at the sex change that alkaline environment carries out template.
On the other hand, above-mentioned renaturation zone is set at the required environment of nucleic acid renaturation, for example is set to satisfy 1) melting temperature(Tm) that is set in nucleic acid following (not heat or cool off), 2 as means) be set at slightly acidic～weakly alkaline (about pH7 ± 3), 3) contain suitable positively charged ion, 4) do not contain the environment of hydrogen bond inhibitor all conditions such as (for example urea and guanidinesalts etc.).For example, because the temperature condition when carrying out nucleic acid renaturation is according to the melting temperature(Tm) of template and primer and different,, be 30～70 ℃ when for example using the primer of 15～30 base pairs so cannot treat different things as the same, good especially among the present invention is 30～40 ℃.Nucleic acid renaturation is meant that mutual complementary single-chain nucleic acid forms two strands, the actual annealing that is meant template and primer of the nucleic acid renaturation under the environment that carries out PCR.
Among the present invention, move to above-mentioned denatured areas by the reaction solution that imports in above-mentioned stream, this reaction solution is exposed to the environment that above-mentioned denatured areas sets, in addition, by moving to above-mentioned renaturation zone, this reaction solution is exposed to the environment that above-mentioned renaturation zone sets.
In addition, among the present invention, above-mentioned denatured areas better is to form with temperature-control device by the outer setting sex change at this stream, makes the reaction solution that moves in stream to be heated to more than the above-mentioned nucleic and melting temperature; Above-mentioned renaturation zone better is to form with temperature-control device by the outer setting sex change at this stream, makes the reaction solution that moves in stream to be heated to below the above-mentioned nucleic and melting temperature.
The used nucleic acid synthetic enzyme of method of the present invention is the enzyme that can be used for nucleic acid amplification, so long as generally the enzyme that can obtain all can use, is not particularly limited, and can exemplify archaeal dna polymerase, ligase enzyme, reversed transcriptive enzyme, RNA polymerase etc. particularly.In addition, also these nucleic acid synthetic enzyme can be used in combination.
Among the present invention, can also use PCR method in the past and ligase chain reaction (LCR) (ligase chainreaction:LCR) method is employed has a stable on heating nucleic acid synthetic enzyme.The nucleic acid synthetic enzyme is fixed in the stream in above-mentioned renaturation zone, and the heating of being carried out in the time of can not standing to make the template sex change etc. do not have stable on heating nucleic acid synthetic enzyme so can use.
Among the present invention, the nucleic acid synthetic enzyme can be useful in the enzyme that has optimum temperuture under 30～40 ℃ the temperature, so can select the enzyme of the less expensive that can't use among in the past the PCR.In addition, can also use other enzyme except that the general kernel acid enzyme simultaneously.Therefore, the enzyme that was difficult to use simultaneously when in the past carrying out PCR, for example correct the enzyme etc. of the mispairing of the nucleic acid that has synthesized, can improve the reliability of amplification than PCR in the past by using simultaneously.
Among the present invention, the enzyme that better is to use the high enzyme of reaction efficiency and obtains easily, particularly, better be to use the reliability of duplicating high derive from colibacillary dna polymerase i.In addition, though the reliability of duplicating is low slightly, also can use dna polymerase i to remove the Ke Lienuo segment at exonuclease activity position (Klenow fragment) etc.
Above-mentioned nucleic acid synthetic enzyme for example can be immobilized in surface of beads and be filled at least a portion of stream in above-mentioned renaturation zone, also can directly be immobilized in the inner wall surface of stream.
When using the nucleic acid synthetic enzyme that is fixed on the bead, fixed nucleic acid synthetic enzyme is contacted efficiently, with reaction solution here, so can improve reaction efficiency.
In addition, the nucleic acid synthetic enzyme is fixed under the situation of inner wall surface in described renaturation zone at least, and the structure of device of the present invention can form simply.That is, when forming such stream, at first can make the nucleic acid synthetic enzyme be immobilized in the whole surface of stream and form overall flow paths, in this way, even the enzyme deactivation of denatured areas, the enzyme in renaturation zone still keeps active condition, so can easily form required stream.
The material of the bead of immobilized nucleic acids synthetic enzyme is not particularly limited, can preferably exemplify metal particle, glass particle, resin granulation etc., good especially latex beads and the immobilization good, enzyme of chitosan bead consistency such and biomolecules of being to use is easy to bead.The size of above-mentioned bead can suitably be set so long as the size that can be filled in the above-mentioned stream gets final product, and general diameter is 0.4～100 μ m, better is that diameter is 1～50 μ m.
In addition, the material that forms above-mentioned stream better is that heat conductivity is higher, and is stable in the required temperature range of PCR, is difficult for being corroded by electrolyte solution and organic solvent, is difficult for absorption nucleic acid and protein.Material with thermotolerance and erosion resistance is except glass, quartz, silicon, can also exemplify various plastics, better be that general be difficult for absorption nucleic acid and the lining of proteinic material are generally acknowledged with polyethylene, polypropylene etc. in surface (inner wall surface that contacts with reaction solution) at them, or import the molecule that polyoxyethylene glycol (PEG) etc. contains a large amount of hydrophilic functional groups with covalent linkage and suppress nucleic acid and absorption of proteins.
The above-mentioned method that the nucleic acid synthetic enzyme is immobilized in the inner wall surface of surface of beads or stream can adopt known method such as carrying method, entrapping method, covalent attachment method, crosslinking, electrostatic adhesion method, owing to can carry out enzyme reaction repeatedly, so covalent attachment method and crosslinking are special ideal.For example, the covalent attachment method can be carried out according to the method for records such as Japanese patent laid-open 3-164177 communique, can (for example import reactive higher functional group in the inner wall surface of surface of beads and stream, chloroformyl (chlorine acyl), carboxyl, amino, thiol group (sulfenyl), epoxy group(ing) etc.), by making this functional group and the carbonyl that is present in the surface of nucleic acid synthetic enzyme, amino, thiol group (sulfenyl) reaction carrying out immobilization.
At least contain template, primer, phosphate cpd and metal ion among the present invention in the employed reaction solution.
Above-mentioned template is the nucleic acid as the amplification target, can use by the natural type of ordinary method preparation or the nucleic acid of non-natural type.The concentration of the above-mentioned template in the reaction solution is preferably 0.01～100pM, more preferably 0.1～10pM usually.
Above-mentioned primer is the nucleic acid that has at least with a part of base sequence complementary base sequence of above-mentioned template, operable nucleic acid gets final product in general PCR method and the LCR method, better be to be designed to increase efficiently, better be to use the sequence of 15～30 bases usually as the nucleic acid of target.For example, can easily make by using the automatic synthesizer of nucleic acid as the nucleic acid of primer.The concentration of the above-mentioned primer in the reaction solution is preferably 0.01～1 μ M usually, more preferably 0.1～0.2 μ M.
In addition, for the detection of back with separate, also contain in the above-mentioned primer by the altered non-natural type of chemically modified nucleic acid.Above-mentioned non-natural type nucleic acid is not particularly limited, and can preferably exemplify nucleic acid oligomer with vitamin H or FITC mark, has the nucleic acid oligomer of phosphorothioate bond and contain the mosaic nucleic acid etc. of PNA (peptide nucleic acid(PNA)) and natural type nucleic acid.
Above-mentioned phosphate cpd is the composition of the substrate during as amplification of nucleic acid, for example use under archaeal dna polymerase and reversed transcriptive enzyme the situation, contain dNTP, be the equal amount of mixture that the mixture of dATP, dCTP, dGTP and dTTP better is to use 4 kinds of deoxynucleotide triphosphoric acids with arbitrary proportion as the nucleic acid synthetic enzyme.On the other hand, use under the situation of ligase enzyme, better be to use NTP, can particularly preferably exemplify ATP and GTP.The concentration of the above-mentioned phosphate cpd in the reaction solution can suitably be set, and better is 0.01～1mM usually, is more preferably 0.1～0.5mM.
Above-mentioned metal ion can exemplify potassium ion (K
+), sodium ion (Na
+), magnesium ion (Mg
2+) etc.By containing such metal ion, can realize stabilization, the enzymic activityization of double-strandednucleic acid, the effects such as fidelity raising of synthetic nucleic acid.The concentration of the above-mentioned metal ion in the reaction solution is generally, and potassium ion and sodium ion better are 10～200mM, are more preferably 50～100mM.In addition, magnesium ion better is 1～5mM, is more preferably 1.5～2.5mM.
In the method for the present invention, better be the liquid supply speed of above-mentioned reaction solution and the conditions such as length of stream suitably adjusted, make the sex change that can in stream, carry out template efficiently, sex change the template and the annealing and the nucleic acid of primer synthetic.These conditions are owing to be subjected to the influence of speed of response etc. of nucleic acid synthetic enzyme of length, the use of length, the synthetic nucleic acid of template, therefore cannot treat different things as the same, common above-mentioned reaction solution is 1～60 second by the above-mentioned denatured areas time once, it better is 5～30 seconds, by the above-mentioned renaturation zone time once be 5～300 seconds, better be 10～120 seconds.
Below, with reference to the accompanying drawings the employed nucleic acid amplifier of nucleic acid amplification method of the present invention is described, use identical symbol with a part in principle, it illustrates omission.
Among Fig. 1, represented a kind of embodiment of nucleic acid amplifier of the present invention.In the nucleic acid amplifier 10, form on the substrate 1 of temperature province B with temperature province A and renaturation in sex change and to have the stream 2 of specifying internal diameter, make it use among the temperature province B alternately circuitous with temperature province A and described renaturation in described sex change, respectively by described sex change temperature province A and temperature province B many times of described renaturation, can make this stream 2 become stream thus with denatured areas and renaturation zone, make above-mentioned nucleic acid denaturation become the reaction of degeneration of strand in the described denatured areas as template, in the described renaturation zone nucleic acid of this formation strand and above-mentioned as primer nucleic acid annealing and carry out the nucleic acid building-up reactions.Be provided with the bead filling part 3 of the bead of filling the nucleic acid synthetic enzyme surface immobilized on the part in the renaturation zone of above-mentioned stream in many places.In addition, the two ends of above-mentioned stream 2 are provided with the taking-up hole 2b that is used in this stream injecting the filling orifice 2a of reaction solution and is used to take out the reaction solution that the amplified reaction of nucleic acid is through with.
In addition, the amplification mode figure of a part that has represented the stream of above-mentioned nucleic acid amplifier among Fig. 2, be filled in the surface immobilized immobilized nucleic acids synthetic enzyme 6 of nucleic acid synthetic enzyme 5 of bead 4 in the above-mentioned bead filling part 3, make the nucleic acid synthetic enzyme 5 that fixedly is formed into immobilized nucleic acids synthetic enzyme 6 to contact with the reaction solution that moves through stream.When being filled into immobilized nucleic acids synthetic enzyme 6 in the stream, better be entrance and exit setting at bead filling part 3 have suitable aperture strainer so that this immobilized nucleic acids synthetic enzyme 6 can not spill.The material of strainer is not particularly limited, and better is to be difficult for absorption nucleic acid, can preferably exemplify Mierocrystalline cellulose etc.
When using this nucleic acid amplifier 10, will contain the direction feed flow of the reaction solution of template, primer, phosphate cpd and metal ion at least along arrow among the figure by outside liquid feed devices (not shown) such as pumps.Thus, described template is after the denatured areas of above-mentioned stream 2 forms strand by thermally denature, carry out in the renaturation zone of above-mentioned stream described single-stranded template and with the annealing reaction of this template complementary primer, carry out complementary strand synthetic of described single-stranded template again at above-mentioned bead filling part 3 by immobilized nucleic acids synthetic enzyme 6, once carry out a PCR circulation by the denatured areas and the renaturation zone of above-mentioned stream respectively.
Among the present invention, above-mentioned stream 2 respectively the sex change by aforesaid substrate with temperature province and renaturation with temperature province once more than, formation gets final product like this, in order to carry out nucleic acid amplification efficiently, better is by 20～40 times.
In addition, the size of above-mentioned stream 2 better be by the aperture is reduced, specific surface area increases makes thermal conduction carry out easily, and do not produce heat fluctuation (with reference to No. 280 5366 volumes of Science (1998), 1046-1048 page or leaf (Kopp MU, Mello AJ, Manz A. work).Flow path width among the present invention is 20～200 μ m, better is 50～100 μ m, and the degree of depth is 20～200 μ m, better is 40～100 μ m.The flow path width of filling the part of immobilized nucleic acids synthetic enzyme 6 in addition is 20～3000 μ m, better is 50～1000 μ m, and the degree of depth is 20～1000 μ m, better is 40～500 μ m.
The material that forms above-mentioned stream 2 better is that heat conductivity is higher, and is stable in the required temperature range of PCR, is difficult for being corroded by electrolyte solution and organic solvent, is difficult for absorption nucleic acid and protein.For example, material with thermotolerance and erosion resistance is except glass, quartz, silicon, can also exemplify various plastics, better be that general be difficult for absorption nucleic acid and the lining of proteinic material are generally acknowledged with polyethylene, polypropylene etc. in surface (surface that contacts with reaction solution) at them, or import the molecule that polyoxyethylene glycol (PEG) etc. contains a large amount of hydrophilic functional groups with covalent linkage and suppress nucleic acid and absorption of proteins.
Substrate with above-mentioned stream can for example be formed as follows.That is, suitably adopt on 1 substrate that constitutes by above-mentioned material by machining etc. and forms ditch, and the method that adheres to other 1 substrate or film is added a cover on described ditch with above-mentioned specified width, which width and degree of depth.
The another kind of embodiment of having represented nucleic acid amplifier of the present invention among Fig. 3.In this nucleic acid amplifier 20, substrate 1a shown in Figure 1 is the dendritic connection of branch other polylith substrate 1b, 1c, 1d, 1e, 1f, 1g.The mode of connection of these substrates is not limited to mode shown in Figure 3, can connect in many ways, and nucleic acid amplification can be carried out efficiently.
Nucleic acid amplifier 20 uses by the 1st reaction solution that contains above-mentioned template at least and contains above-mentioned primer at least, the reaction solution that the 2nd reaction solution of phosphate cpd and metal ion constitutes, by outside liquid feed devices 11 such as pumps, at first described the 1st reaction solution and described the 2nd reaction solution supply to substrate 1a from the 1st reaction liquid bath 14 and the 2nd reaction liquid bath 15 respectively, the reaction solution that has passed through described substrate 1a by outside liquid feed device 12 directly as template supplying substrate 1b, 1c, 1d, 1e, 1f, 1g, simultaneously, supply with described the 2nd reaction solution from the 2nd reaction liquid bath 15 for reaction substrates such as the primer that consumes in the reaction that replenishes described substrate 1a and phosphate cpds.
And the reaction solution that has passed through aforesaid substrate 1b, 1c, 1d, 1e, 1f can directly reclaim and carry out purification of nucleic acids, also can be connected to the polylith substrate as required and carries out nucleic acid amplification.
In the present embodiment, be provided with stream 7,8 and pump 13, the reaction solution that is used for that a part passed through the reaction solution of aforesaid substrate 1a and has passed through aforesaid substrate 1g utilizes as above-mentioned the 1st reaction solution again.By such stream of feed flow again is set, can prevent that not only template from using up, stably carry out the successive nucleic acid amplification, can also lower running cost.
Be like this under the situation that branch connects substrate dendriticly, better be at each section at least one substrate, for example divide the substrate (substrate 1a) support the front and have on the substrate with path (substrate 1g) that the reaction solution that will pass through substrate utilizes as the 1st reaction solution again the nucleic acid synthetic enzyme that the reliability of fixedly duplicating is high.Thus, can correctly carry out the amplification of template, so even repeat also amplification template correctly of PCR.
The reaction solution circulation and the structure of the circulation stream by denatured areas in the circulation stream and renaturation zone have alternately been represented to be used in the nucleic acid amplifier of the present invention to make at Fig. 4 (a) with (b).
In the circulation stream shown in Fig. 4 (a), form the circulation stream, can control by the outside liquid feed device 13a that the control reaction solution flows to the feed flow of the stream 2c of branch by the stream 2c of specified location ramose branch at stream 2.The reaction solution that enters the stream 2c of branch at the branching portion of stream sex change with temperature province A by the denatured areas in the circulation stream, get back to renaturation temperature province B from the portion of compiling of stream, can pass through again in the renaturation zone in having passed through circulation stream once.
In addition, shown in Fig. 4 (b), above-mentioned circulation stream also can form stream 2 ring-type and not have branch's stream., be arranged on ring-type stream 2 midway here as the supply of reaction solution, the reaction liquid bath 16 that takes out part, the reaction solution that imports from described reaction liquid bath 16 by outside liquid feed device 13a along the direction of arrow the figure at stream 2 internal recycle.
In above-mentioned circulation stream, circulate, alternately carry out nucleic acid amplification reaction repeatedly by reaction solution by denatured areas in the above-mentioned circulation stream and renaturation zone, the resulting amplified production of result can be gathered from described reaction liquid bath 16 in Fig. 4 (b) from the outlet collection of stream in Fig. 4 (a).
In the nucleic acid amplifier of the present invention, shown in Fig. 5 (a), the sex change of aforesaid substrate can form by aforesaid substrate 1 for example is set in temperature-control device 33 with temperature province with temperature province and renaturation, and described temperature-control device 33 has with dividing plate 38 separations and has the structure that sex change is used the thermostatic bath 32 of temperature control unit 35 with the thermostatic bath 31 and the renaturation of temperature control unit 34.In the above-mentioned thermostatic bath,, stirrer 36 and 37 are set respectively in order to stir medium, the maintenance temperature homogeneous in the thermostatic bath.
In addition, shown in Fig. 5 (b), can laminated polylith substrate 1, in configuration sex change between each substrate or between the several piece substrate with temperature control unit 39 and renaturation with temperature control unit 40, the sex change that forms aforesaid substrate is with temperature province and renaturation temperature province.
Above-mentioned sex change can keep homo(io)thermism with temperature control unit and above-mentioned renaturation by any temperature-control device with temperature control unit, can preferably exemplify electrical heating element, thermostat, nichrome wire, heating lamp etc. particularly.In addition, above-mentioned sex change can not disposed with substrate contacts ground with temperature control unit with temperature control unit and above-mentioned renaturation.
Another embodiment of having represented nucleic acid amplifier of the present invention among Fig. 6.This nucleic acid amplifier 50 uses 2 kapillaries 51 as stream, kapillary 51 shape in the shape of a spiral curls to be arranged on and has sex change with temperature province A and renaturation in the temperature-control device 52 with temperature province B, make its alternately by described sex change with temperature province and renaturation temperature province.
As shown in Figure 7, the direct immobilized nucleic acids synthetic enzyme 5 of the inner wall surface of above-mentioned kapillary 51.
Above-mentioned material capillaceous is not particularly limited, the material that forms it better is that heat conductivity is higher, stable in the required temperature range of PCR, be difficult for being corroded by electrolyte solution and organic solvent, be difficult for absorption nucleic acid and protein, for example can exemplify glass, plastics etc., better be that polyethylene is used on surface (surface that contacts with reaction solution) at them again, polypropylene etc. are generally acknowledged general be difficult for absorption nucleic acid and the lining of proteinic material, or import the molecule that polyoxyethylene glycol (PEG) etc. contains a large amount of hydrophilic functional groups with covalent linkage and suppress nucleic acid and absorption of proteins.
In addition, constitute employed material capillaceous and also can be semipermeable, have the polymer of prevention by seeing through low molecular character, at this moment the solution that contains low-molecular-weight substrate (for example dNTP and NTP etc.) by use is as the medium that this thermostatic bath capillaceous is set, can also be in kapillary supply response substrate constantly.Semipermeable kapillary can preferably exemplify the tubular fibre that the レ of Mitsubishi one ョ Application and eastern レ etc. sell.
Among the present invention, above-mentioned specification capillaceous is, external diameter 100～1000 μ m better are 200～500 μ m, and internal diameter 20～600 μ m better are 50～150 μ m.
The nucleic acid synthetic enzyme can get final product at whole inner wall surface fixed nucleic acid synthetic enzyme capillaceous with carrying out with the same method of above-mentioned immobilized nucleic acids synthetic enzyme to the immobilization of above-mentioned inner wall surface capillaceous.Under the situation of whole inner wall surface immobilized nucleic acids synthetic enzyme capillaceous, the nucleic acid synthetic enzyme that is immobilized in denatured areas can not exert an influence to the nucleic acid building-up reactions because of inactivations such as heating usually, have active getting final product so be immobilized in the nucleic acid synthetic enzyme in renaturation zone, do not have the problem in the actual use.
If adopt this capillary pipe structure, then can save the filling bead, only in the operation of privileged site immobilized nucleic acids synthetic enzyme, make and also become easy.
Embodiment
Below, the present invention is described in detail to exemplify embodiment, and scope of the present invention is not limited to these
Embodiment.
＜embodiment 1 〉
(making of nucleic acid amplifier)
For as above-mentioned just Fig. 1 and the illustrated ground of Fig. 2, obtain in a stream, being arranged alternately the structure in a plurality of denatured areas and renaturation zone, be formed as follows the stream substrate, in the described stream substrate, with a zone of substrate as the sex change temperature province, and another zone makes a circulation a stream to be configured in the surface as the renaturation temperature province, makes it alternately by these zones.
That is, by with the polyethylene injection moulding, make the laminal substrate (vertical 35mm * horizontal 70mm) of thick 1mm, form at substrate surface by machining have the width shown in the table 1 and the degree of depth, successive ditch in the longitudinal direction, as the stream substrate.At this moment, will have the circuit portion in an adjacent denatured areas and a renaturation zone as a unit.
[table 1]
| Width (μ m) | The degree of depth (μ m) | Length (mm) | |
| Denatured areas part in channel unit | 200 | 200 | 12 |
| Renaturation area part (removing the bead filling part) in channel unit | 200 | 200 | 25 |
| Bead filling part in channel unit | 1000 | 200 | 25 |
The mode chart of the ditch of a channel unit as shown in Figure 8.Here, the sex change of substrate is 12mm with the length as the bank stream of denatured areas among the temperature province A, and the renaturation of substrate is 50mm with the length as the bank stream in renaturation zone among the temperature province B.In addition, the ditch width of the wide diameter part 3a of the stream of filling immobilized nucleic acids synthetic enzyme is 1000 μ m, and the ditch width of other parts is 200 μ m.Constituting by machining formation on the above-mentioned polyethylene substrate in of the ditch series connection of length direction successive ditch by 40 unitary above-mentioned channel units.
On the other hand, the immobilized nucleic acids synthetic enzyme of wide diameter part 3a that is filled in the stream of aforesaid substrate is done following adjustment.
That is, with the chitosan bead carrier (trade(brand)name " キ ト パ one Le BCW-3001 " of 1g (weight in wet base) median size 100 μ m; Fuji Spinning Co., Ltd.'s system) at 5ml PBS damping fluid (137mM NaCl, 8.1mMNa
2HPO
4, 2.68mM KCl, 1.47mM KH
2PO
4, pH7.2) in 4 ℃ of following balances 8 hours.By removing by filter the PBS damping fluid, add 2ml 2.5% glutaraldehyde water solution, activate 2 hours down at 4 ℃.Then, behind elimination 2.5% glutaraldehyde water solution, bead is washed 3 times with 5ml PBS damping fluid.Then, after the elimination PBS damping fluid, add 0.1 μ g/ μ l archaeal dna polymerase Klenow segment ( カ ラ バ イ オ Co., Ltd. the system)/PBS damping fluid of 1ml, reacted 2 hours down, carry out immobilization at 4 ℃ as enzyme solution to bead.The elimination enzyme solution after 5ml PBS damping fluid washing 3 times, is adjusted to 50% pulp-like with the PBS damping fluid.
Bead filling part at above-mentioned stream substrate splashes into, fills the synthetic like this immobilized nucleic acids synthetic enzyme of 2.5 μ l with each channel unit of micropipet.At this moment, can think that the immobilized nucleic acids synthetic enzyme has accounted for only about half of in the bead filling part volume.
The configuration Peltier's element is as temperature control unit on the surface of the subtend on the surface of above-mentioned stream substrate formation ditch.That is, will be separately positioned on certain area of substrate on the surface of stream substrate with the required Peltier's element of temperature province with the required Peltier's element of temperature province with the renaturation of certain area of substrate as 94 ℃ sex change as 37 ℃.
In order to add a cover as stream on the ditch of above-mentioned stream substrate, folded another piece polyethylene substrate of going up is also used the clip clamping, as the nucleic acid amplification reaction substrate.
In addition, will obtain above-mentioned nucleic acid amplifier from joining on the pipe connecting that is connected with the stream inlet part of substrate with above-mentioned nucleic acid amplification reaction with the feed pipe of pump as the high-speed liquid chromatography of liquid feed device.
＜embodiment 2 〉
(nucleic acid amplification reaction)
Use the nucleic acid amplifier of making among the embodiment 1 to carry out the PCR reaction.At this moment, will contain the aqueous solution of following content as its reaction solution.
Reaction solution:
Template double-stranded DNA (73 pairs of bases) 10pM
Forward primer DNA (18 bases) 1 μ M
Reverse primer DNA (18 bases) 1 μ M
DATP, dGTP, each 5 μ M of dCTP, dTTP
MgSO
4 10mM
Dithiothreitol (DTT) 0.1mM
Tris-HCl(pH7.2，25℃) 50mM
Dna sequence dna:
The normal chain of template double-stranded DNA: sequence numbering 1
The minus strand of template double-stranded DNA: sequence numbering 2
Forward primer DNA: sequence numbering 3
Reverse primer DNA: sequence numbering 4
In addition, use in above-mentioned reaction solution, do not contain template double-stranded DNA (73 pairs of bases) solution in contrast.
Reaction solution or contrast solution after 2 minutes, are cooled to 37 ℃ 94 ℃ of following sex change in advance, with liquid feed device liquid supply speed feed flow with 1 μ l/min in the stream of the nucleic acid amplifier of the foregoing description 1.Here, taken the part in the renaturation zone if consider the immobilized nucleic acids synthetic enzyme, the ratio that then is configured in the shared part in renaturation zone and denatured areas in 1 unitary stream internal volume of the stream on the stream substrate of nucleic acid amplifier of the foregoing description 1 is about 7: 1.Thus, can think and 40 the PCR circulations that have been equivalent to repetition of the reaction solution that passed through 40 unitary streams or contrast solution comprise 94 ℃ reaction of degeneration in 30 seconds, 3 minutes 30 seconds annealing/extensions of 37 ℃ in each circulation.
Reaction solution and nucleic acid molecular weight mark in 3% sepharose (TAE damping fluid: 40mM Tris, 19mM acetic acid, 1mM EDTA) the electrophoresis of the part that will pass through the reaction solution of 40 unitary streams or contrast solution before in the stream that imports nucleic acid amplifier dyes the gel that obtains with the 0.5 μ g/ml ethidium bromide aqueous solution.Photo when having represented UV (302nm) irradiation among Fig. 9.Among the figure, 1 is the interior electrophoretic swimming lane of reaction solution before of the stream that imports nucleic acid amplifier, 2 for having passed through the electrophoretic swimming lane of reaction solution of 40 unitary streams, and 3 is the electrophoretic swimming lane of nucleic acid molecular weight mark (50bp ladder), and 4 for having passed through the electrophoretic swimming lane of contrast solution of 40 unitary streams.
As shown in Figure 9, the interior contained template double-stranded DNA (73 pairs of bases) of reaction solution before of stream that imports nucleic acid amplifier is micro-, is not detected (swimming lane 1).In addition, do not detect DNA (swimming lane 4) from the contrast solution that has passed through 40 unitary streams yet.On the other hand, gone out DNA near the position detection of reaction solution 70～75 pairs of base length of nucleic acid molecular weight mark mobility (swimming lane 3) of having passed through 40 unitary streams, confirmed that the template double-stranded DNA (73 pairs of bases) in the reaction solution is amplified (swimming lane 2).
[sequence table]
Sequence numbering 1: as the normal chain of the template double-stranded DNA of 73 base length of PCR reaction template
Sequence numbering 2: as the minus strand of the template double-stranded DNA of 73 base length of PCR reaction template
Sequence numbering 3: the PCR forward primer DNA that is used for amplification template DNA
Sequence numbering 4: the PCR reverse primer DNA that is used for amplification template DNA
The possibility of utilizing on the industry
What the present invention can be used in template nucleic acid etc. copies amplification efficiently.
Claims (9)
1. nucleic acid amplifier, it is to have at least one stream in inside, in this stream, flow through the reaction solution of the nucleic acid, nucleic acid, phosphate cpd and the metal ion that contain at least as template as primer, in this stream amplification of nucleic acid nucleic acid amplifier
It is characterized in that, described stream has denatured areas and renaturation zone simultaneously, carry out the reaction of degeneration that the two strands of the intramolecularly of described nucleic acid as template and/or intermolecular formation is unwind in the described denatured areas, described two strands is unwind in the described renaturation zone forms two strands as the nucleic acid of template and described nucleic acid as primer, and the nucleic acid synthetic enzyme is fixed in described renaturation zone.
2. nucleic acid amplifier as claimed in claim 1, its feature also are to have the temperature control unit that can heat above-mentioned denatured areas and above-mentioned renaturation zone be remained on the temperature lower than above-mentioned denatured areas.
3. nucleic acid amplifier as claimed in claim 1 or 2, its feature are that also described nucleic acid synthetic enzyme is fixed on the bead, and this bead is filled in the described renaturation zone at least.
4. nucleic acid amplifier as claimed in claim 1 or 2, its feature are that also described nucleic acid synthetic enzyme is fixed on the inner wall surface in described renaturation zone at least.
5. as each the described nucleic acid amplifier in the claim 1～4, its feature also is, described denatured areas and described renaturation zone alternatively are set in described stream.
6. as each the described nucleic acid amplifier in the claim 1～5, its feature is that also described nucleic acid synthetic enzyme has optimum temperuture in 30～40 ℃ temperature range.
7. as each the described nucleic acid amplifier in the claim 1～6, its feature also is to have the circulation stream, and should the circulation stream has the structure of described renaturation zone and described denatured areas.
8. as each the described nucleic acid amplifier in the claim 1～7, its feature also is also have the liquid feed device that the control reaction solution flows to, and the flow direction of this liquid feed device control reaction solution periodically reverses.
9. nucleic acid amplification method, it is to be used for containing nucleic acid as template, the reaction solution amplification of nucleic acid, phosphate cpd and metal ion as primer at least as the method for the nucleic acid of template, it is characterized in that, comprise: the denaturing step that the two strands of the intramolecularly of described nucleic acid as template and/or intermolecular formation is unwind; (b) with step (a) different zone, make that this two strands that obtains in the step (a) unwind as the nucleic acid of template and described as forming double-stranded renaturation step between the nucleic acid of primer; (c) in step (b) and/or after, make to be fixed in the step that the nucleic acid synthetic enzyme zone that comprises the zone of carrying out step (b), that keep active condition contacts with reaction solution.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP273430/2003 | 2003-07-11 | ||
| JP2003273430 | 2003-07-11 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1820067A true CN1820067A (en) | 2006-08-16 |
| CN100434502C CN100434502C (en) | 2008-11-19 |
Family
ID=34056021
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004800196435A Expired - Fee Related CN100434502C (en) | 2003-07-11 | 2004-07-12 | Nucleic acid amplifier and method of nucleic acid amplification |
Country Status (6)
| Country | Link |
|---|---|
| US (2) | US20060257878A1 (en) |
| EP (1) | EP1659170A1 (en) |
| JP (1) | JPWO2005005594A1 (en) |
| CN (1) | CN100434502C (en) |
| HK (1) | HK1092175A1 (en) |
| WO (1) | WO2005005594A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107271240A (en) * | 2017-07-07 | 2017-10-20 | 黑龙江然得基尔医学科技发展有限公司 | A kind of colouring method of cell DNA |
| CN109266516A (en) * | 2018-09-28 | 2019-01-25 | 中国科学院长春光学精密机械与物理研究所 | The production method and detection device of DNA cloning device, DNA cloning device |
| CN113528333A (en) * | 2021-07-20 | 2021-10-22 | 北京擎科生物科技有限公司 | Nucleic acid synthesis reaction apparatus and nucleic acid synthesis method |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007126565A (en) * | 2005-11-04 | 2007-05-24 | Dainippon Toryo Co Ltd | Coating composition for galvanized steel sheet or zinc alloy-plated steel sheet and steel sheet coated therewith |
| CN101589157B (en) * | 2006-10-06 | 2014-04-02 | 万达利亚研究公司 | Method foe a continuous rapid thermal cycle system |
| JP2009268432A (en) * | 2008-05-09 | 2009-11-19 | Canon Inc | Method for measuring target nucleic acid |
| RU2675502C1 (en) * | 2013-11-29 | 2018-12-19 | Конинклейке Филипс Н.В. | Optical control of chemical reaction |
| EP3296386B1 (en) * | 2015-05-12 | 2020-05-06 | Xiamen University | Nucleic acid amplification reaction tube capable of controlling liquid circulation path |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4683195A (en) * | 1986-01-30 | 1987-07-28 | Cetus Corporation | Process for amplifying, detecting, and/or-cloning nucleic acid sequences |
| GB8917963D0 (en) * | 1989-08-05 | 1989-09-20 | Scras | Apparatus for repeated automatic execution of a thermal cycle for treatment of biological samples |
| JPH0630776A (en) * | 1992-07-13 | 1994-02-08 | Rikagaku Kenkyusho | Method for amplifying dna and device for amplifying dna |
| JPH0690756A (en) * | 1992-09-10 | 1994-04-05 | Fuso Yakuhin Kogyo Kk | Two stage thermocycle pcr method |
| US5863801A (en) * | 1996-06-14 | 1999-01-26 | Sarnoff Corporation | Automated nucleic acid isolation |
| WO1998022625A1 (en) * | 1996-11-20 | 1998-05-28 | The Regents Of The University Of Michigan | Microfabricated isothermal nucleic acid amplification devices and methods |
| EA004327B1 (en) * | 1999-03-19 | 2004-04-29 | Такара Био. Инк. | Method for amplifying nucleic acid sequence |
| JP4162187B2 (en) * | 2001-12-11 | 2008-10-08 | 宮城県 | Method and apparatus for quantifying pyrophosphate and nucleic acid |
-
2004
- 2004-07-12 US US10/564,060 patent/US20060257878A1/en not_active Abandoned
- 2004-07-12 WO PCT/JP2004/009942 patent/WO2005005594A1/en active Application Filing
- 2004-07-12 EP EP04747408A patent/EP1659170A1/en not_active Withdrawn
- 2004-07-12 JP JP2005511563A patent/JPWO2005005594A1/en active Pending
- 2004-07-12 CN CNB2004800196435A patent/CN100434502C/en not_active Expired - Fee Related
-
2006
- 2006-11-17 HK HK06112676.0A patent/HK1092175A1/en not_active IP Right Cessation
-
2008
- 2008-01-24 US US12/018,905 patent/US20080139408A1/en not_active Abandoned
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107271240A (en) * | 2017-07-07 | 2017-10-20 | 黑龙江然得基尔医学科技发展有限公司 | A kind of colouring method of cell DNA |
| CN109266516A (en) * | 2018-09-28 | 2019-01-25 | 中国科学院长春光学精密机械与物理研究所 | The production method and detection device of DNA cloning device, DNA cloning device |
| CN109266516B (en) * | 2018-09-28 | 2022-04-05 | 中国科学院长春光学精密机械与物理研究所 | DNA amplification device, method for manufacturing DNA amplification device, and detection device |
| CN113528333A (en) * | 2021-07-20 | 2021-10-22 | 北京擎科生物科技有限公司 | Nucleic acid synthesis reaction apparatus and nucleic acid synthesis method |
Also Published As
| Publication number | Publication date |
|---|---|
| HK1092175A1 (en) | 2007-02-02 |
| WO2005005594A1 (en) | 2005-01-20 |
| EP1659170A1 (en) | 2006-05-24 |
| US20060257878A1 (en) | 2006-11-16 |
| CN100434502C (en) | 2008-11-19 |
| JPWO2005005594A1 (en) | 2006-10-26 |
| US20080139408A1 (en) | 2008-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2432040T3 (en) | Nucleic acid amplification with continuous flow emulsion | |
| CN1299419A (en) | Compositions and method for enhanced synthesis of nucleic acid molecules | |
| CN1248781C (en) | Device for heat-dependent chain amplication of target nucleic acid sequences | |
| US20140256568A1 (en) | Sample multiplexing | |
| CN1942590A (en) | Fluidic devices and methods for multiplex chemical and biochemical reactions | |
| JP2019515674A (en) | Immobilization-based systems and methods for genetic analysis and other applications | |
| CN1820067A (en) | Nucleic acid amplifier and method of nucleic acid amplification | |
| JP2003299485A (en) | Temperature control-type microreactor and microreactor system | |
| JP2017506877A5 (en) | ||
| JP7296409B2 (en) | Compositions for use in polynucleotide sequencing | |
| US6204025B1 (en) | Efficient linking of nucleic acid segments | |
| WO2013028643A1 (en) | Preparation of polynucleotides on a solid substrate for sequencing | |
| CN101078024A (en) | Method and apparatus for concentrating and amplifying nucleic acid in single micro chamber | |
| CN111183144A (en) | Click-based connection | |
| Khilko et al. | DNA assembly with error correction on a droplet digital microfluidics platform | |
| CN1834261A (en) | Gene typing chips, prepn. process and uses thereof | |
| CN1196755A (en) | Methods for determining coat colour genotype of pig | |
| CN1140632C (en) | Methods for DNA amplification and kits therefor | |
| US20140087955A1 (en) | Devices and methods for efficient capture of nucleic acids | |
| Li et al. | Melting analysis on microbeads in rapid temperature-gradient inside microchannels for single nucleotide polymorphisms detection | |
| CN1200115C (en) | Oligonucleotide chip and its application of detecting muatatonal site of hepatitis B virus | |
| CN101063159A (en) | Systemic displacement polymerase lymerase chain reaction | |
| CN1405325A (en) | RNA probe preparation method, Target nucleic acid detection method and RNA probe preparation kit | |
| CN1291017C (en) | Novel gene P40 containing acyl group carrying protein-like structural domain | |
| EP4293124A2 (en) | Combined solution phase and solid phase dna amplification |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1092175 Country of ref document: HK |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| REG | Reference to a national code |
Ref country code: HK Ref legal event code: GR Ref document number: 1092175 Country of ref document: HK |
|
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20081119 Termination date: 20100712 |